Two mammalian proteins important for analysis of in vitro DNA replication have been characterized. These proteins are DNA alpha-polymerase and helix-destabilizing protein-1 from mouse myeloma. Discrete fragments of the helix-destabilizing protein were produced by controlled proteolysis with trypsin. These fragments were purified and used in studies on structure-function relationships. We found that the N-terminal region of native helix-destabilizing protein-1 is not required for DNA binding and that DNA binding by the native protein occurs without significant protein:protein cooperativity. The kinetic mechanism of alpha-polymerase was studied using a simplified replication system in which the enzyme acted in a strictly processive fashion. The experimental results were consistent with a rapid equilibrium-random Bi Uni Uni Bi ping pong mechanism. It was also found that the initiation phase of the alpha polymerase reaction could be studied independently of the elongation phase.